Method of enhancing the action of a medicament

ABSTRACT

This invention is concerned with a method of prolonging the action of a medicament in a mammal by administering parenterally thereto an emulsion comprising an aqueous phase dispersed substantially uniformly throughout a metabolizable vegetable, animal or hydrocarbon oil containing one or more hydrocarbons of about 12 to 18 carbon atoms, the aqueous phase having an average particle size of about 0.1 to 10 microns and including an effective dose of a medicament. The medicament may be therapeutic or prophylactic in nature, and more specifically, may include live or killed microorganisms, pollen extract and a host of other materials. In another aspect of the invention non-specific resistance of the mammal is induced by administering parenterally the aforedescribed emulsion in which the medicament is a lipopolysaccharide and such an emulsion induces in the mammal resistance to radiation, shock and infection when subjected thereto. In a specific sense the fetus of a mammal may derive higher resistance to infection or radiation by administration to the female mammal the aforedescribed emulsion. The oil phase of the emulsion decreases the LD50 of a toxic medicament. The mammals in which such responses can be achieved are mice, rabbits, monkeys, rats, guinea pigs, pigs, dogs, and the like.

1s] 3,67,19 45] July 1, i972 [72] Inventor:

Samuel J. Prlgal, 225 West 106th St., New York, NY. 10025 [22] Filed:Jan. 26, 1970 [211 Appl. No.: 5,950

Related US. Application Data [63] Continuation-impart of Ser. No.490,642, Sept. 27,

I965, Pat. No. 3,492,399, which is a continuation-inpart of Ser. Nos.170,242, Jan. 31, 1962, abandoned, and Ser. No. 401,669, Oct. 6, 1964,abandoned, which is a continuation-in-part of Ser. No. 47,364, Aug. 4,1960, abandoned, and Ser. No. 28,011, May 10, 1960, Pat. No. 3,096,249.

521 US. Cl. ..424/8, 424/7, 424/88,

424/89, 424/91, 424/92, 424/180 51 Int. Cl. ..A6lk 27/12 [58] Field orSearch ..424/88-91, 7, 8

[56] References Cited UNITED STATES PATENTS 3,096,249 7/1963 Prigal..424/3'5 x 3,149,036 9/1964 Woodhour et a1. ...424/88 x 3,185,6255/1965 Brown, ...424/22 x 3,384,544 5/1968 Walton et al ..424/89 x3,399,263 8/1968 Strazdins et a1 ..424/88 3,492,399 1/1970 Prigal ...42491 3,531,565 9/1970 Webb et al ..424/92 OTHER PUBLICATIONS Loveless J.Immunol. 79(l):68 79 July 1957 Repository Im- Treatment of Pollinosis byMeans of a Single Injection of Emulsified Extract V. Preparation,Standardization and Administration Brown Review of Allergy and AppliedImmunology 14:302- 347 May- June 1960 Opsiphylactic Injection Treatmentof Grass Hay Fever and Pollen Asthma Prigal New York State J. Med6l:3,6l 6- 3,623 Nov. 1961 Improved Emulsions of Allergens With Built-InSafety Features Primary Examiner-Shep K. Rose Attorney-Donald P.Gillette [57] ABSTRACT This invention is concerned with a method ofprolonging the action of a medicament in a mammal by administeringparenterally thereto an emulsion comprising an aqueous phase dispersedsubstantially uniformly throughout a metabolizable vegetable, animal orhydrocarbon oil containing one or more hydrocarbons of about 12 to 18carbon atoms, the aqueous phase having an average particle size of about0.1 to 10 microns and including an efi'ective dose of a medicament. Themedicament may be therapeutic or prophylactic in nature, and morespecifically, may include live or killed microorganisms, pollen extractand a host of other materials.

In another aspect of the invention non-specific resistance of the mammalis induced by administering parenterally the aforedescribed emulsion inwhich the medicament is a lipopolysaccharide and such an emulsioninduces in the mammal resistance to radiation, shock and infection whensubjected thereto. In a specific sense the fetus of a mammal may derivehigher resistance to infection or radiation by administration to thefemale mammal the aforedescribed emulsion.

The oil phase of the emulsion decreases the LD of a toxic medicament.The mammals 1n wh1ch such responses can be achieved are mice, rabbits,monkeys, rats, guinea pigs, pigs, dogs, and the like.

6 Claims, No Drawings METHOD OF ENHANCING THE ACTION OF A MEDICAMENTBACKGROUND OF THE INVENTION This application is a continuation-in-partof my co-pending application Ser. No. 490,642 filed Sept. 27, 1965, nowU.S.

. Pat, No 3,492,399 which was a continuation-in-part of my ap- FIELD OFTHE INVENTION This invention relates to methods of prolonging the actionof medicaments through prolonged release by having such medicamentcontained within an aqueous phase dispersed in a vegetable, animal orhydrocarbon oil containing about 12 to 18 carbon atoms. The same actionscan be employed to induce non-specific resistance in a mammal throughthe prolonged release of a toxicant, such as a lippolysaccharide.

BACKGROUND OF THE INVENTION In the production of immunity in mammals,whether against infectious diseases or in the treatment of allergicdisorders, the use of the water-in-oil type emulsion with the oil phaseof the present invention as an adjuvant, has proved remarkablyeffective. Antibody production of a higher order and of prolongedduration can be achieved in this fashion, as exemplified by theadministration of influenza vaccine, in which multiple aqueousinjections provide immunity for about one year, whereas the same vaccinegiven as an emulsion provides at least nine years protection. Weakantigens administered in this fashion havealso been shown to possess anincreased level of effectiveness.

These and the other objects of this invention are accomplished byemployment of a water-in-oil emulsion comprising an aqueous phasedispersed substantially uniformly throughout a metabolizable vegetable,animal or hydrocarbon oil including one or more hydrocarbons containingbetween about 12-18 carbon atoms, the aqueous phase having an averageparticle size of about 0.1 to microns, and containing a prophylactic.-or therapeutic agent which does not produce hypersensitivity.

The therapeutic and prophylactic agents of this invention useful inmammals are those which induce prolonged therapy or prolonged immunity.This is a most significant feature of this invention. Illustrative ofsuch therapeutic and prophylactic agents are the agents which can beemployed for the purpose of this invention and which are not soluble inthe oil phase. The agent may be insoluble in water or completely solubletherein or it may be intermediate of such solubilities, but it cannot besoluble in oil, otherwise it will not be entrapped as part of theaqueous phase within the oil phase. The agent may include allergens,e.g., house dust extract (Center), hoiise dust extract (Endo)lyophilized or aqueous, tree pollen, timothy pollen, plantain pollen,ragweed pollen, molds, epidermals, etc.; poison ivy; anesthetics, e.g.,procaine, hydrochloride, etc.; antibiotics, e.g., procaine potassiumpencillin G (suspension), tetracycline, chloramphenicol,oxytetracycline, nitrofuran, etc.; antimethabolites antihistamines,e.g., diphenydramine hydrochloride, tripelennamine hydrochloride;chlorprophyenpyridamine, etc.; antienz yme, e.g., enostigmine, etc.;anticoagulent, e.g., heparin, etc.; antiinflammatory agent, e.g.,phenylbutazone, 3-5-dioxo-l, 2- diphenyl-4-n-butyl pyrazolidine, etc.;bronchodilators, e.g., epinephrine, isopropylarterenol,ethylnorepinephrine hydrochloride, protokylol hydrochloride, etc.;coagulant, e. g., medadione sodium bisulfite, etc.; diuretics, e.g.,mercumatilin, mercaptomerin, sodium meralluride-theophylline, etc.;enzymes, e.g., chymotrypsin crystallized trypsin, etc.; hormones, e.g.,insulin, globulin insulin, sodium succinate ester of hydrocortisone,ACTH (corticotrophin), corticotropin zinc, methicortelone,methylprednisolone acetate, pituitrin, etc.; stimulants, e.g., caffeinesodium benzoate, nikethamide, etc.; tranquilizers, e.g., chlorpromazine,methaminodiazepoxide, etc.; vaccines, e.g. typhoid (triple), influenza,polimyelitis, staphylococcus toxoid, etc.; vitamins, e.g., ascorbicacid, vitamin B complex, vitamin 8, vitamin B, etc. The quantity ofagent which is incorporated in the aqueous phase may vary considerablyin view of the nature of the material employed and the purpose of usebut, in general, the amount employed may be about 1.5 to 10,000 timesgreater or even higher than is used conventionally for purposes ofinoculation.

Within the scope of this invention and forming an embodiment thereof isa method for inducing non-specific host resistance by the parenteraladministration of an emulsion, as

defined hereinbefore, wherein said emulsion contains an cffective doseof a lipopolysaccharide, so that the host upon exposure to radiation,shock (traumatic, hemorrhagic, allergic, gravitational, vibrational, andburn), and infection (bacterial, rickettsial, protozoan, viral naturallyand artificially encountered), exhibits resistance thereto.

As has been discussed above, the water-in-oil emulsion of this inventionenable the administration of relatively toxic doses of allergins tohypersensitive mammals with safety. The same modality can therefore beapplied to primary toxic agents, such as bacteria] endotoxin, so thatpresumably lethal doses do not kill when injected. Furthermore, abundantevidence is available that lipopolysaccharides (LPS) derived frombacterial endotoxin induce in experimental animals multiple favorableresponses, enabling them to withstand a variety of noxious challenges asdiscussed hereinabove. The following examples demonstrate the inductionof resistance in mice against a lethal Staphylococcus infection, Sarcoma(180) implant and against a whole-body X-irradiation.

EXAMPLE I 88 parts of n-hexadecane, 10 parts purified mannide monooleateand 2 parts glyceryl mono-oleate were combined. An aqueous phase wasprepared by suspending solid LPS derived from E. coli 026:B6 andprepared by the Bouivin method in sterile pyrogen-free distilled waterin a concentration of 20 and 40 mg/ml. To each ml. was added one drop(0.5cc) of FeCl-,,(l0ercent). The aqueous LPS suspension was emulsifiedwith the oil mixture under sterile conditions in batches of 6-8 ml.,using equal portions of the oil and aqueous phases. 5 minutes of shakingin an electrical shaker which moves in three distinct planes, at about3,000 rpm, was used for emulsification.

When the LPS is emulsified, it is possible to give 24 times the LD dose,without killing, for prolonged effect. (This is demonstrated fullyhereinafter). Thus, 2mg. of LPS as a parenteral aqueous injection kills100 percent of all mice to which it is administered within 72 hours.However, when the same dose is emulsified, as above, upon injection 100percent survival can be achieved. Mammals thus treated can withstandchallenges against Sarcoma implant, irradiation and infection. Forexample, in one study 1 mg. of emulsified LPS injected sub-cutaneouslyinto 60 mice and challenged at 2, 14, 21, 36 50 and 70 days with alethal staphylococcus injection of the protective LPS emulsion yieldedsurvivors ranging from 90-20 percent with an over-all percent survivalof 53.8 percent. In sharp contrast, 30 mice unprotected by the LPSshowed no survivors when challenged by the staph infection. In anotherstudy of 60 mice in which 2 mg. of LPS was injected sub-cutaneously asan emulsion, the percent survival, when challenged with a staphinfection, was 46.6 percent. In both cases, the control group showedpercent mortality.

LPS treated mice were also challenged with a Sarcoma implant, whichnormally kills 8090 percent of the animals so treated. The LPS protectedgroup, however, showed survival rates ranging from 30-80 percent, andthis type of resistance was detectable for as long as 92 days after theinitial LPS injection.

Yet another group of mice protected by LPS was exposed to lethalwhole-body radiation (680r). These mice resisted the lethal action andincreased survival was noted for a minimum of 23 days, with the survivalrate being 20 percent at 2 days (post LPS parenteral injection), 29percent at 5 days, 33 percent at 14 days and 27.8 percent at 23 days. Inmarked contrast, the survival rate of untreated mice exposed to the sameradiation dose was only 16.6 percent.

A related embodiment and one which demonstrates a significant advantageof this invention, is a method for decreasing the LD of a therapeutic orprophylactic agent which comprises the incorporation of said agent inthe metabolizable oils of this invention as a waterin-oil emulsionwherein the aqueous phase contains 0.1- micron-size particles. Throughsuch incorporation, it is possible to 'utilize prophylactic ortherapeutic agent materials which heretofore were too toxic to beemployed in sufficiently large dosage unit form to achieve a desiredeffect without endangering the mammal. Exemplary of agents which areparticularly important in this regard are the pharmaceutically importantpolysaccharides (zymosan, glucan and bacterial endotoxins orlipopolysaccharides). Also exemplary are large dosages of such commonallergens as extracts of the pollen or ragweed, timothy and plantain,house dust and epidermals administered in dosage units which wouldusually be toxic unless emulsified in the above-described hydrocarbonoils.

Utilization of the novel emulsions of this invention produces anincreased repository effect which results in prolongation of the actionof the therapeutic or prophylactic agent contained therein. For example,lipopolysaccharide (LPS), even if given in less than LD dose, stillexerts some toxic effect and the sum total of its therapeutic benefit isonly for short duration a matter of several days to a week. On the otherhand LPS given 2-4 times the LD in the form of a water-in-oil emulsionof this invention, exerts prolonged effect against a Sarcoma implant(over 90 days). Against an intravenous injection oflive staphlococci, aneffect for 77 days is observed, and over 23 days against lethalwhole-body radiation. These results are achieved, furthermore, withoutkilling, despite the administration of doses which are normally lethaldoses. Therefore, another embodiment of this invention is a method forachieving such prolongation of therapeutic effect, comprising theadministration to the mammal of a water-in-oil emulsion, as definedhereinbefore.

In connection with the production of resistance through utilization ofthe foregoing LPS emulsions, a method for producing resistance of thefetus to infection or radiation by administration parenteral to thepregnant female mammal of an emulsion of this invention containing aneffective amount of a lipopolysaccharide forms an embodiment of thisinvention. This embodiment is of great significance, for example, in thediagnosis or treatment of pregnant female mammals where use of any formof radiation has, in the past, been limited in view of the unusualsensitivity of the fetus to radiation.

Another embodiment of this invention comprises a method for modifyingand purifying pollen antigens by the incorporation of an effectiveamount, generally about 0.05-0.0 ml. ofa solution of heavy metal salt,generally up to about 10 percent by weight, such as iron salts, as forexample, ferric chloride, into each ml. of allergen extract in anemulsion of this invention, wherein the therapeutic or prophylacticagent contained therein is pollen extract. Current methods ofhyposensitization of pollenosis, wherein there is sensitivity to variouspollens, the classic example of which is ragweed hayfever, continue tobe inadequate, presumably due to the interference with antibodyproduction by some of the extraneous allergens of the pollen.

In connection with this embodiment, the pH of the aqueous phase issignificant and it is preferred to achieve a pH of the allergen extractson the acid side. For example, in the range of between about a pH of l-5and preferably 2-4. Therefore, another embodiment of this invention is amethod for modifying and purifying pollen antigens comprisingacidification of a water-in-oil emulsion, as defined hereinabove, toachieve the foregoing pH ranges. Within the scope of this embodimentalso is the novel modified pollen antigen produced by the ferricchloride in the foregoing manner.

It has been the practice to immunize some allergic mammals by a seriesof sub-cutaneous injections, beginning with small doses of allergen andgradually increasing the dosage at weekly intervals, until satisfactoryimmunization was achieved. Usually it requires approximately 20injections to immunize a mammal against hay fever or asthma due topollen, dust or mold sensitivity. The administration of the allergen orantigen may produce untoward effects, if care is not exercised inregulating the quantity of the antigen. In the case of aqueous extractsof antigens, immediate reactions, such as hay fever (allergic rhinitis),hives, asthma and/or shock, may occur, if the quantity of the antigenexceeds the tolerance of the mammal.

Through extensive investigations, it has been determined that thedangers which may arise from wrong diagnosis of a mammals tolerance canbe substantially eliminated by using the allergen in a form which willbe released in small quantities at a slow rate at the site of injection.Actually, the rate of release and assimilation is such that the mammalneed only be given a single injection (or a few injections), in contrastto the 20 or so injections now being widely used. The technique by whichthis revolutionary change is made possible will be discussed in greaterdetail hereinbelow.

In accordance with the present invention, the allergen is admixed with apolyvalent metallic ion in quantities sufficient to combine chemicallywith at least part of the proteinaceous material contained therein; andthen the allergen thus treated is emulsified within a non-toxic oilofthis invention.

In a more particular aspect of the present invention, the metallic ionis admixed with the allergen in a quantity greater than that required tocombine chemically with all of the proteinaceous material and the excessis usable as an indicating material to designate the state ofemulsification, thus indicating whether it is safe to use.

Still another embodiment of this invention is a method for producingspecific immunity by the administration of the emulsion of thisinvention wherein the therapeutic agent is a live, virulent orattenuated organism.

In reference to the use oflive microorganisms in emulsions, it is knownthat killed vaccines do not always produce immunity. On the other hand,immunity may be achieved by the use of live organisms, usuallyattenuated. This creates in turn the possibility of overwhelming thehost with infection. When, however, these live organisms are given as awater-in-oil emulsion for slow release, it is possible to produceimmunity without overwhelming the defenses of the mammal. In essence,this is how most immunity is achieved, by natural exposure to smalldoses of live organisms which may produce subclinical disease andimmunity. Thus, a method for producing specific immunity by theparenteral administration of live organisms as the water-in-oil emulsionof this invention forms an embodiment of this invention. In general, thenatural virulent form of the organism is preferred, although attenuatedand even the killed organism can also be employed. In the case of thekilled organism, a more effective and prolonged immunity can beachieved.

This embodiment thus is concerned with a water-in-oil emulsion whereinthe dispersed aqueous phase contains a microorganism, which for thepresent purpose may mean in a generic sense killed, attenuated or avirulent organism. in a more particular sense, the emulsion can containa polyvalent metal cation which serves to enhance its stability. Theadjuvant oil, as defined herein, exerts a therapeutic effect by virtueof the effect it has on the reticulo endothelial system, (abbreviated asthe REShereinafter). This is a surprising beneficial effect, becausewhen the RES is stimulated, the response to an antigen for developmentofspecific immunity is greater than would be otherwise.

The discovery that the oil has a therapeutic effect through itsstimulation of the RES is important, because it points to the utility ofusing emulsions for immunologic purposes. The physical makelup of theemulsion demonstrates the uniqueness of this approach, because eachglobule or particle of the dispersed phase is surrounded by oil. Beforethe aqueous phase is reached at the site of injection of the emulsion,the oil must be removed by metabolism or by phagocytosis, thus the RESis being stimulated in all likelihood just prior to the time that theantigen is absorbed. Thus a double-barteled effect is created, with theresult that the oil cooperates or synergizes with the antigen. Thisunique effect may not be observed if the emulsion is not stable, becauseat the site of injection the antigen and oil are being absorbedsimultaneously. It is very desirable to have the RES stimulated first,because the immunologic response is far greater to a specific antigenfor the development of specific immunity.

To derive the maximum benefit of having the RES stimulated beforeattemptingto establish specific immunity, an injection oflipopolysaccharide such as bacterial endotoxins or zymosan from yeast,etc., could be employed before administering the antigen. The parenteralinjection for stimulation of the RES can be made about 2 to 6 days priorto the injection for developing specific immunity.

The aqueous phase of the emulsion includes water, and the antigen, withor without the non-toxic polyvalent metal cation mentioned previously.As to the source of the polyvalent metal cation, generally ionizablesalts are employed, and these may be salts ofsuch metals as calcium,magnesium, aluminum,

zinc, copper, iron, cobalt, nickel, mercury, etc. The metal salts maybe, for example, chlorides, nitrates, sulfates, acetates, benzoates,bitartrate, biphosphate, borate, bromide, butyrate, carbonate, citrate,hydroxide, iodide, isobutyrate, isovalerate, lactate, linolate, malate,randalate, oleate, oxalate, phosphate, propionate, salicylate, stearate,succinate, tartrate, etc. Specific examples are nickel acetate, cupricacetate, magnesium b enzoate, magnesium biphosphate, aluminum borate,ferric'bromide, ferric calcium butyrate, ferric citrate,magnesiumcitrate, cobalt citrate, aluminum hydroxide, ferrous iodide,cupric oleate, ferrous phosphate, magnesium phosphate, calciumpropionate, mercuric salicylate, mercuric stearate, ferric tartrate,cupric tartrate, etc.

The polyvalent metal ionic material may also service as an indicatingmaterial for determining by spot testing the state of emulsification ofthe final composition. By virtue of the metal salt being dispersedproperly in the emulsion, it cannot be detected by standard proceduresof spot testing. But, if the dispersed phase should agglomerate or theemulsion is not made properly, then the uncombined metal salt willreadily react with a suitable analytical reagent to produce a coloredproduct. Specific combinations of metal salt and reagent are, forexample, aluminum chloride and other aluminum salts with alizarin orquinalizarin; calcium nitrate or other calcium salts with dihydroxytartaric acid-osazone or glyoxalbis (2- hydroxyanil); copper nitrate orcopper sulfate with'o-toldine and ammonium thiocyanate or rubeanic acidor alizarine blue or diphenyl carbazone, or zinc diethyldithiocarbarnate; ferric chloride or other iron salts with potassiumferrocyanide or ammonium thiocyanate or potassium thiocyanate or,'-dipyridyl or disodium-l, 2 dihydroxy benzene-3, 5 disulfonate;magnesium chloride or magnesium nitrate with quinalizarin; mercuricchloride or mercuric nitrate with cuprous iodide; zinc nitrate or zincchloride with potassium ferricyanide and pphenetidine; cobalt nitratewith chromatropic acid dioxime or sodium pentacyano piperidine ferroate;nickel nitrate or chloride with dimethyl glyoxime or rubeanic acid; etc.

Since the metal salts can be detected in very minute concentrations, forexample, 1 part of metal salt in 200,000-5,000,000 parts of water, it isnot necessary to have a large quantity to be detected. Generally, themetal salts may be present in the final composition in such smallconcentrations as about 1 to 5 parts per million (abbreviated ppm"). Themetal salt may be as high as 5,000 ppm or even higher; however, it isdesirable to maintain the amount as low as possible and yet beconsistent with good detection. Usually, about to 3,000 ppm of metalsalt will serve to provide good detection. The method of detecting themetal salts is described in Feigl's book entitled Spot Tests inlnorganicAnalysis," 5th edition, Elservier Publishing Co., New York, I958, andsuch disclosure is hereby incorporated by reference. One excellentmethod of detection for ferric chloride is to saturate filter paper withpotassium ferrocyanide (0.02 normal) and then add a drop of the'finalcomposition to the paper. If the characteristic blue color does notappear immediately, then it can be said that the composition is safe foruse. Ammonium thiocyanate is 2 percent solution may be used instead ofpotassium ferrocyanide, giving a characteristic rusty or brown reactionproduct with the ferric ion.

The dispersed phase of the dispersion or emulsion is comprised ofparticles or globules having an average size of about 0.1 to 10 microns.The size of particle of globule will deter mine to some extent thestability of the dispersion or emulsion. Consequently, for relativelystable dispersions or emulsions, the average particle or globule size isabout 0.1 to 2 microns, whereas, for relatively faster rates of release,the average size may be about 2 to 10 microns. By virtue of having theparticles or globules surrounded by an oily medium, the antigen isreleased for absorption at a rate dependent upon the rate of oilabsorption or metabolization. Consequently, in the present invention,there is an interplay of several factors which causes a slow andsubstantially constant rate of release and slow absorption of antigen,namely, (1) the stability of the emulsion or dispersion, and (2) therate of absorption or metabolization os the oil material.

The dispersed phase of the emulsion may also contain an additionalindicating material which serves to designate the state ofemulsification. The additional indicating material may be a non-toxicdye or pigment. Such an indicating material must not be soluble in theoil phase, but should be soluble in the dispersed aqueous phase. Thechemical nature of the indicator is not material, i.e., it can be anyorganic or inorganic material, but the concentration in which it is usedmust not be toxic to the subject being inoculated.

Various dyes, such as the aeridine dyes, azo dyes, indigoid dyes,pthalein dyes, triphenylmethane dyes, etc., can be used. Specificexamples of the medicinal dyes are acriflavine, methylene blue, Congored, Evans blue, scarlet red, indigo, tyrian purple, fiuroscein,indopthalein, merbromin, phenolpthalein, phenolsulphonthalein,sulfobromothalein, fuchsin, methyl rosanaline, etc. Specific examples ofthe F. D. & C. certified dyes, are blue No. l (brillant blue), blue No.2 (indigo carmine), green No. l (guinea green), green No. 2 (light greenS F yellowish), green No. 3 (fast green FCF), orange No. 1 (orange l),red No. l (ponceau 3R), red No. 2 (amaranth), red No. 3 (erythrosine),red No. 4 (ponceau 5X), yellow No. l (naphthol yellow), yellow No. 2(naphthol yellow K salt), yellow No. 5 (tetrazine), yellow No. 6 (sunsetyellow FCF), etc. The indicating material may also be an indicator whichproduces color at a given pH level. If the pH of the emulsion does notcause the indicator to be colored, then the state of emulsification canbe determined by having some of the emulsion containing the indicatorcontact filter paper or the like saturated with an acid or base, as thecase may be. If the emulsion is proper, color will not appearimmediately, even though the indicator would normally be colored uponcontacting the acid or base. Specific examples of the indicators are:

Methyl Violet 6B Metacresol Purple Metanil Yellow Methyl Red ChlorphenolRed Bromcresol Purple Direct Purple Metacresol Purple Alizarin YellowG.G. Alizarin Yellow R Tropaeolin Sodium alizarin sulfonate BromcresolGreen The indicating material may comprise from about .00001 to 0.5percent, more usually about 0.00001 to 0.005 percent, by weight of thedispersed phase. At relatively low concentrations, an emulsioncontaining the dispersed phase of globules ofless than about microns,particularly less than 2 microns, will give little or no evidence ofcolor. If color is apparent, it is present uniformly throughout theemulsion, making no distinction that it is actually present only in thedispersed phase. When the globules grow larger in size, through crackingof the emulsion, color will reappear. It has also been found, quiteunexpectedly, that some indicating materials, when they are surfaceactive, such as, for example, amaranth and phenolsulfothalein, promoteemulsification and/or stabilization of the emulsion.

It has also been observed that the combination of a surface activeindicating material, such as, for example, amaranth and/orphenolsulfopthalein, with the non-colored indicating material or metalsalt, such as, for example, the ferric salt, e.g., ferric chloride,promotes emulsification and stabilizes the emulsion beyond what would beexpected from their individual effects. For reasons not clearlyunderstood, these materials synergize to give an improvement which isnot forseeable on the basis of their expected properties. The relativeamounts of the two indicating materials may vary within the ranges givenhereinabove for each type.

Aside from the emulsifying effect which is obtained from the indicatingmaterial, it is also contemplated to employ an emulsifying agent oragents for this type of emulsion. The emulsifying agent must benon-toxic when used in the amounts at which emulsification is produced.Various classes of emulsifiers may be used for this invention includingsuch materials as a partial ester of a polyhydric alcohol and a fattyacid, a glycolether, long chain fatty alcoholpolyoxyethylene, long chainfatty acid polyoxyethylene, a partial ester of a fatty acid and apolyhydric alcohol condensed with ethylene oxide, etc. All theemulsifiers are either amohoteric or non-ionic in character. The partialesters usually contain at least two free hydroxyl groups; consequently,they are derived from polyhydric alcohols containing at least threehydroxyl groups. These alcohols may contain as high as 12 hydroxylgroups, although usually those containing up to six hydroxyl groups areused more often. The fatty acid, employed to esterify part of thehydroxyl groups, may contain about eight to 24 carbon atoms, moreusually about 12 to 18 carbon atoms. The polyhydric alcohols are, forexample, glycerol, pentaerythritol, mannitol, etc. The fatty acids are,for example, lauric acid, oleic acid, stearic acid, octanoic acid, etc.Specific examples of such emulsifiers are mannide mono-oleate, glycerylmono-oleate, mannitol mono-oleate, glycerol monooleate, pentaertyritolmono-laurate, glucose dioctanoate, etc. The glycol ethers are derivedfrom alkylene glycol and a long chain fatty alcohol, e.g., thosecontaining about 12 to 24 carbon atoms in the alcohol molecule. Theglycol ethers may have the formula:

wherein R may be an alkyl group of about 24 carbon atoms or hydrogen ora hydroxy substituted alkylene group, or hydroxy substituted alkylgroup, and R may be an alkyl group of about 12 to 24 carbon atoms. Thecondensate of ethylene oxide and a fatty alcohol may be represented bythe following formula:

R0 (CH,CH 0),,OH wherein R is an alkyl group containing about 12 to 24carbon atoms and n" is an integer ranging from about 10 to 40. Thecondensate of ethylene oxide and a fatty acid may be represented asfollows:

wherein R is an alkyl group containing from about 8 to 24 carbon atoms,preferably 12 to 18 carbon atoms and n" is an integer of 10 to 40.

The emulsifier is usually employed in an amount of about l to 15 percentby volume based on the dispersed phase. The amount of emulsifieremployed may vary with the type of emulsifier being used. Allemulsifiers are not equivalent in their effectiveness; i.e., some arebetter than others.

It has also been found that the combination of a partial estercontaining five free hydroxyl groups, such as mannitol monooleate, witha partial ester containing two free hydroxyl groups, such as glycerolmono-oleate, produces an unusually effective combination of emulsifiers,as evident from the longer stability of the emulsion upon standing andfrom the fact the emulsion is obtained faster. Generally, about 1 to 10parts by volume of the partial ester containing five free hydroxylgroups are admixed with 1 to 10 parts by volume of the partial estercontaining two free hydroxyl groups.

The microorganism to be emulsified can be alive, modified, attenuatedand/or killed. The microorganism can be bacterial, viral and/orrickettsial. Specific examples of microorganisms are poliomyelitisvirus, measles virus, yellow fever virus, influenza viruses, adenoviruses, common cold viruses, black leg virus, hog cholera virus,renderpest virus, fowl pow virus, newcastle virus, distemper virus,infectius canine hepatitis, coryne bacterium diptheriae, clostridiumtetani, hemophilus pertussis, mycobacterium tuberculosis, brucella,bacterium tulerense, salmonella paratyphosa, salmonella typhosa, choleravibrio, mocrococcus pyogenes var. aureus (staphylococcus),streptococcus, etc. The microorganisms may be used singly in theemulsions or in various combinations of two or more of the same. Forexample any two or more of microorganisms which are responsible fordiptheria, tetanus, pertussis, polio and measles. Similarly, adeno andcold viruses may be combined.

By utilizing the microorganisms in emulsion form several importantadvantages are realized, through the delayed absorption, namely, lessmaterial is required of the vaccine, longer or prolonged immunity isinduced, less untoward or unfavorable reactions are encountered and theimmunity approximates natural immunity to a greater extent than has beenpossible by vaccines heretofore.

The oil portion of the emulsion may vary considerably in amount relativeto the aqueous phase. In general, the aqueous phase may comprise about0.1 to 2.5 parts by volume per part by volume of oil. In the case ofemulsions made with vegetable oils, the aqueous phase is preferablyabout 0.1 to 1.5 parts by volume per part of oil. The singlehydrocarbons are preferably emulsified with relatively larger quantitiesof aqueous material, namely, about 0.5 to 2.5 parts by volume of aqueousphase per part of oil.

The preparation of the emulsion may involve combining an emulsifier or acombination of emulsifiers with the oil, adding the antigen andindicator to the aqueous material and then combining the two resultantphases to produce the emulsion. The emulsion can be obtained by varioustechniques, such as by mechanical shaking at speeds of about 2,000 rpmand higher, by ultra-Sonics, by a colloid mill, by stirring and/or by adouble syringe method involving two syringes which are interconnected bya double-hubbed needle, the size of which is EXAMPLE lll InfluenzaVaccine Polyvalent n-hexadecane (.88 part) Mannide mono-oleate (.l pan)Glyceryl mono-oleate (.02 part) Particle size (average) lcc lcc

The stability of the emulsion shown in Example IV was remarkably betterthan an emulsion which did not contain FeCl EXAMPLE V Killed PolioVaccine (Types 1, 2 and 3 USPH Standards) n-hexadecane (0.88 part)Mannide mono-oleate (0.06 part) Glyceryl mono-oleate (0.06 part) EXAMPLEVl Live measles virus* (lzlO dilution) n-hexadecane (088 part) Mannidemono-oleate (0.06 part) Glyceryl mono-oleate (0.06 part) Ice Ice

Attenuated in accordance with procedure of Frankell et al Fed. Proc.,Vol. 17, page 551 (I958) The aforementioned formulas and the emulsionsof this invention are useful for developing immunity, and in particular,they can be used in mammals such as, for example, mice, rabbits,monkeys, rats, guinea pigs, dogs, etc. to ascertain the effects ofvarious microorganisms in developing specific and nonspecificimmunologic responses.

Although the foregoing discussion has been directed to the use of asingle hydrocarbon oil adjuvant, included within the scope of eachembodiment is the use of such hydrocarbons in admixture with a suitablevegetable oil, or other suitable metabolizable non-hydrocarbon oil. lnmany cases, such admixtures are preferred because of the extremely lowincidence of tissue irritation exhibited thereby, upon parenteraladministration. Varying mixture proportions can be used depending uponthe nature of the desired emulsion. These mixtures can be produced invarious ways. For example, the vegetable oil and metabolizablehydrocarbon e.g. n-hexadecane, can be mixed with suitable amounts of anemulsifier. In general, the emulsion is made with n-hexadecane and thevegetable oil, together with its emulsifier, is used to dilute the outeroily phase. Various proportions of each can be used to bring about adesired effect. Or, separate emulsions can be made, each containing theactive ingredient, and thereafter mixed in suitable proportions. Thislatter technique permits some of the material to be absorbed morequickly via the vegetable oil, while the agent admixed with thehydrocarbon is absorbed more slowly.

Another embodiment of this invention is a method for enhancing theadjuvant effect of vegetable oil emulsions, which comprises adding tothese emulsions increasing concentrations of n-hexadecane or the otheremulsion oils of this invention. The following example more fullydemonstrates this embodiment.

EXAMPLE Vll To a mixture of 0.88 part n-hexadecane, 0.1 part mannidemono-oleate and 0.02 part glyceryl mono-oleate is added 2-3 ml.influenza polyvalant vaccine in a peanut oil emulsion comprising 0.88part peanut oil, 0.1 part mannide mono-oleate and 0.02 part glycerylmono-oleate. This emulsion, upon subcutaneous administration to a mouse,favorably modifies the inflammatory response and yet produces enhancedanti-body protection, since comparable vegetable oils are such pooradjuvants and this is overcome by the added n-hexadecane.

What is claimed is:

l. In a method for prolonging repository effect immunity or therapyinduced by an oil-insoluble medicament in a mammal which comprises:administering parenterally to such mammal an in ectab e, stable,homogeneous w/o emulsion; said emulsion comprising 0.l to 2.5 parts byvolume, per part of oil phase emulsified with mannide mono-oleate andglycerol monoleate, of an aqueous dispersed phase containing awatersoluble color indicator designating the state of emulsificiationand the prolonged repository effect immunity-inducing ortherapy-inducing oil-insoluble medicament dispersed as globules orparticles substantially uniformly through an oil phase selected from thegroup consisting of animal, vegetable, and a hydrocarbon containingabout l2l 8 carbon atoms, the improvement comprising the step ofeffecting a slow and substantially constant rate of release and slowabsorption thereof of said globules of aqueous dispersed phase by virtueof having each globule prepared and determined by color indicator tohave an average particle size of about 0.1 to 10 microns.

2. A method of inducing non-specific resistance in a mammal by theparenteral administration of the emulsion of claim 1 in which themedicament is a lipopolysaccharide.

3. A method for increasing the LD of a toxic medicament in a mammal bythe parenteral administration to said mammal of the emulsion of claim 1.

4. A method for producing resistance of a fetus or neonate of a mammalto radiation by parenteral administration to the female mammal carryingsaid fetus or neonate the emulsion of claim 2.

5. A method for producing specific immunity in a mammal by theparenteral administration of the emulsion of claim 1 in which themedicament is selected from the group consisting of live virulent andattenuated microorganisms which produces the disease against whichimmunity is sought and the killed microorganisms of the same.

6. The method of claim 1 wherein emulsion is further characterized byhaving included therewith about 0.5 to 0.1 m1. of a 10 percent by weightsolution of ferric chloride and the medicament is an allergen usedtherapeutically, said allergen beingan inhalant, mold, epidermal,pollen, or house dust extract allergen.

2. A method of inducing non-specific resistance in a mammal by theparenteral administration of the emulsion of claim 1 in which themedicament is a lipopolysaccharide.
 3. A method for increasing the LD50of a toxic medicament in a mammal by the parenteral administration tosaid mammal of the emulsion of claim
 1. 4. A method for producingresistance of a fetus or neonate of a mammal to radiation by parenteraladministration to the female mammal carrying said fetus or neonate theemulsion of claim
 2. 5. A method for producing specific immunity in amammal by the parenteral administration of the emulsion of claim 1 inwhich the medicament is selected from the group consisting of livevirulent and attenuated microorganisms which produces the diseaseagainst which immunity is sought and the killed microorganisms of thesame.
 6. The method of claim 1 wherein emulsion is further characterizedby having included therewith about 0.5 to 0.1 ml. of a 10 percent byweight solution of ferric chloride and the medicament is an allergenused therapeutically, said allergen being an inhalant, mold, epidermal,pollen, or house dust extract allergen.